Quantification & Dilutions II Assignment - BIO 140L PDF

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Summary

Please review MicroLecture Module 7, Lab Manual Chapter 6, Food Safety Spinach Procedure MP4, & Food Safety After Incubation MP4....

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Name(s):

Kaylee Shrewsbury

Quantification Assignment Please see Quantification Results video (MP4) and refer to Quantification Results Colony Counts for all Groups Excel file (Module 4). Additionally, reference Lab Manual: Chapter 5 Quantification of Microorganisms (pg. 9) and Photo Atlas (pgs. 283-285). Complete the table. Calculate the OCD for Groups # 1- #6 of Plates D-F and the average OCD for all six groups. Provide OCD values in Exponent form! I included my work at the end. Group #

OCD (CFU/mL)

1 2

3.05 x 10^8 CFU/mL

3

2.87 x 10^8 CFU/mL

4

2.82 x 10^8 CFU/mL

5 6

2.75 x 10^8 CFU/mL 3.30 x 10^8 CFU/mL

AVERAGE OCD

2.97 x 10^8 CFU/mL

3.0 x 10^8 CFU/mL

1. Using the spread plate procedure, explain why an OCD value for a group is smaller than the average OCD. A group might have a smaller OCD value than the average due to many possible experimental errors. For example, they could have set the pipette to a smaller measurement than it was supposed to be and added too little to the tubes. Additionally, when dispensing the volume into the tubes, they could have not released all of the volume. They could have also forgotten to change tips in between pipetting, which then causes contamination. They could have also vortexed each tube too much.

2. Using the spread plate procedure, explain why an OCD value for a group is larger than the average OCD. A group might have a larger OCD value than the average due to many possible experimental errors as well. They could have not set the pipette to the right measurement and added too much volume. When dispensing the volume into the tubes, they could have hit the plunger too hard going past the threshold and releasing too much volume into the tube. They could have also forgotten to change 1

tips in between pipetting causing contamination and extra volume in the tube. They also could have not vortexed each tube or not vortexed them enough to mix the volume together.

3. Name one real-life situation, excluding those mentioned in the Chapter 5 Quantification, in which we would want to know bacterial counts. One example of a real-life situation that was excluded from those mentioned in the Chapter 5 Quantification would be when a doctor is prescribing medication such as antibiotics. A doctor needs to know the bacterial count in order to prescribe the right dosage. It can be harmful if the dosage is too high for the actual number of bacteria in the patient. They are able to dilute the medication in order for it to treat the right number of bacteria.

4. What is the advantage of the spread plate method over direct count and turbidometric count methods? Explain fully. There are many advantages of using the spread plate method over direct count and turbidometric count methods. The spread plate method allows you to count the cells directly on a culture plate. It is an easy technique that is inexpensive since it only requires agar plates and dilution buffer solution. This method only counts live bacteria since dead cells will not grow on the plates. It is very accurate for bacteria at a wide range of concentrations. The direct count method is able to show both dead and live bacteria and there must be at least 10^7 organisms per milliliter before they can be seen on the slide. The turbidometric method requires a spectrophotometer and cannot distinguish between dead or alive bacteria.

5. What is the estimated concentration (CFU/mL) of cells, based on observations that the buffer solution became clear in Tube B (Lab Manual Chapter 5, pg. 9) and the calculated Average OCD? Show your calculations. Average OCD x TDF of Tube B 2.97 x 10^8 CFU / mL x 10^-2 = 2.97 x 10^6 CFU / mL There can be 10^6 organisms in the clear buffer in tube B. The drawing is shown below.

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Dilution Problems II Please note that for the dilution problems use only the following diluent volumes (0.9 mL, 9 mL or 9.9 mL). Please see Chapter 5 Quantification (Module 4) and Photo Atlas pgs. 283-285. Answers should be in terms of EXPONENTS. -6

1. Draw TWO ways of making 10 dilution. Indicate all transfer volumes and diluent volumes. Include IDF & TDF values. Can provide digital drawings or hand-drawings (insert photos/scans)

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2. One mL of a sample that had been diluted to 10-8 was spread on TSA plate. The plate yielded 80 CFU. What is the original cell density (OCD)? Show calculation & provide appropriate units. OCD = CFU / mL = # colonies / TDF x Plate volume 80 CFU / (10^-8 x 1mL) = 8.0 x 10^9 CFU / mL

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3. 0.1 mL of a sample that had been diluted to 10 was spread on TSA plate. The plate yielded 150 CFU. What is the original cell density (OCD)? Show calculation & provide appropriate units. OCD = CFU / mL = # colonies / TDF x Plate volume 150 CFU / (10^-7 x .1mL) = 1.50 x 10^10 CFU / mL

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